Site-specific recombinase technology

Site-specific recombinase technologies are genome engineering tools that depend on recombinase enzymes to replace targeted sections of DNA.

In the late 1980s gene targeting in murine embryonic stem (ES-)cells enabled the transmission of mutations into the mouse germ line and emerged as a novel option to study the genetic basis of regulatory networks as they exist in the genome. Still, classical gene targeting proved to be limited in several ways as gene functions became irreversibly destroyed by the marker gene that had to be introduced for selecting recombinant ES cells. These early steps led to animals in which the mutation was present in all cells of the body from the beginning leading to complex phenotypes and/or early lethality. There was a clear need for methods to restrict these mutations to specific points in development and specific cell types. This dream became reality when groups in the USA were able to introduce bacteriophage and yeast-derived site-specific recombination (SSR-) systems into mammalian cells as well as into the mouse

Common genetic engineering strategies require a permanent modification of the target genome. To this end great sophistication has to be invested in the design of routes applied for the delivery of transgenes. Although for biotechnological purposes random integration is still common, it may result in unpredictable gene expression due to variable transgene copy numbers, lack of control about integration sites and associated mutations. The molecular requirements in the stem cell field are much more stringent. Here, homologous recombination (HR) can, in principle, provide specificity to the integration process, but for eukaryotes it is compromised by an extremely low efficiency. Although meganucleases, zinc-finger- and transcription activator-like effector nucleases (ZFNs and TALENs) are actual tools supporting HR, it was the availability of site-specific recombinases (SSRs) which triggered the rational construction of cell lines with predictable properties. Nowadays both technologies, HR and SSR can be combined in highly efficient "tag-and-exchange technologies".

Many site-specific recombination systems have been identified to perform these DNA rearrangements for a variety of purposes, but nearly all of these belong to either of two families, tyrosine recombinases (YR) and serine recombinases (SR), depending on their mechanism. These two families can mediate up to three types of DNA rearrangements (integration, excision/resolution, and inversion) along different reaction routes based on their origin and architecture.

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